| Backgroundlntracerebral hemorrhage is a common cerebro vascular disease. Epidemiological studies suggest that the incidence of intracerebral hemorrhage range from60to80/100000, accounting for18.8-47.6%of acute cerebrovascular disease. In all types of cerebrovascular disease, intracerebral hemorrhage is one of cerebrovascular diseases with the highest risk in mortality and cripple. About35-52%of patients died within30days and only20%of them achieved self-care ability in six months.Although people have made great progress in the basic research and in the clinical treatment of intracerebral hemorrhage, the prognosis of patients has not been significantly improved for a long time.Many studies confirmed that the destruction of the blood-brain barrier after intracerebral hemorrhage was a common and important pathophysiological change.The incidence and development of cerebral hemorrhage after Intracerebral hemorrhage are the main reason leading to patients serious defect of neurological function, hernia and even death. The blood-brain barrier, composed of brain capillary endothelial cells and their tight junction and endothelial basement membrane and astrocyte end feet, is the important structure for the maintenance and regulation of the neural microenvironment and is the prerequisite and basis of the central nervous system for physiological activity. While endothelial cells and its tight junctions are the basic structures. In Structure, it is a complex of a set of protein molecular element. These protein components including:transmembrane protein, mainly by occludins, claudins, junction associated molecules (JAM) protein family composition; subsidiary cytoplasmic protein, mainly by the zonula occludens (ZO) protein family. Membrane components of tight junctions molecules interact with each other and with their neighbors on tight junctions membrane protein polymerization, to format a stable, tight junctions between cells. In the numerous blood-brain barrier tight junction of molecular cells, caudins is the main component of the tight junction protein molecular cells. It participates in maintaining close connection of selective permeability and cell polarization.Claudin-5is the specific protein of a brain microvascular endothelial cell tight junction.Ohtsuki were found by culturing rat brain microvascular endothelial cells and the exogenous expression of claudin-5could induce the formation of blood brain barrier.ZO-1is the first component that was confirmed to be closely connected to protein and MAGUK. ZO is mainly composed of PDZ domain, SH3(Src homologous3)domain and GUK domain. ZO-1has the function of maintaining epithelial cell polarity and is indirectly involved in the formation of cytoskeleton and cell tight junctions and it is one of the important component of the proteins. Besides, ZO-1plays the role of signal transduction in barrier function and metastasis of cancer cells. The various factors that cause the destruction of blood-brain barrier structure and function Is the main reason leading to vasognic cerebral edema after intracerebral hemorrhage and development. Li Bing injected autologous blood into rat brain and found that occludin decreased along with permeability of BBB increasing. After the incidence of intracerebral hemorrhage,inflammatory cytokines, hemoglobin decomposition products, of thrombin, oxidative stress, complement and matrix metalloproteinases all can destroy BBB.Hemoglobin is the main component of red blood cells in the blood and is a protein for carrying oxygen in high organisms. In human’s body, the hemoglobin is mainly composed by2asubunits and2βsubunits. Each subunit is composed by a globin and a heme molecule. When intracerebral hemorrhage happens, the blood enters the brain through broken blood vessel and formats to hematoma. The hemoglobin is one of main components for the formation of hematoma and it is released by the disintegration of a large number of red blood cells. Studies show that the hemoglobin released from the disintegration of red blood cells after intracerebral hemorrhage happening is one of the main factor that leads to vasogenic brain edema after intracerebral hemorrhage. Xi injected intact cells3d in rat brains, and he found the permeability of brain blood barrier and formation of brain edema blood appeared. While injecting lytic red blood cells, he found the increase of permeability of brain blood barrier and the formation of brain edema within24hours. It indicates that after intracerebral hemorrhage, intact red blood cells will not cause brain edema, while the hemoglobin released from the disintegration of red blood cells may cause brain edema after intracerebral hemorrhage. In the research of hemoglobin oxygen-carrying vector, Randal found after a short time of pulmonary vascular endothelial cell Hb in vitro, it can induce production of endothelial cell action stress fibers and formation of cell gap, as well as enhance of the permeability of bovine pulmonary vascular endothelial cells. The evidence of the research from Silva shows that after Hb, it can be worked as mediators of inflammation to induce change of endothelial cell cytoskeletal. These studies shows that Hb can directly role in endothelial cells and induce damage of tight junctions and increase of permeability.Rho kinase is a serine/threonine protein kinase. It is widely expressed in various tissues. There are two basic isoforms (Rho kinase Ⅰ and Rho kinase Ⅱ),and The Rho kinase Ⅱ is highly expressed in the brain. Rho kinase substrate includes myosin light chain phosphatase (MLCP), myosin, ERM (ezrin/radaxin/moesin), LIM kinase and cofilin Various factors result in Rho kinase activation and myosin light chain (MLC) phosphorylation and cause a variety of biological effects. For example, contraction of vascular smooth muscle cell, cell adhesion and migration, formation of stress fiber and cytoskeleton reorganization. Therefore, Rho kinase activated by many factors resulted in MLC phosphorylation and caused a variety of biological effects.Myosin light chain is constituted by the basic light chain and regulatory light chain. It is considered that Myosin is mainly responsible for regulating the cytoskeleton cells and participating in a variety of physiological activities. These functions are relative to the myosin light china phosphorylation and dephosphorylation The myosin light china phosphorylation is both regulated by myosin light chain kinase(MLCK) and myosin light chain phosphatase (MLCP). Both of them regulate the dynamic balance between MLC phosphorylation and non-phosphorylation. MLCK is activated by the upstream calcium-calmodulin complex and causes the increase in p-MLC. After phosphorylation, MLCP results in inactivation. The inactive MLCP can not be dephosphorylation and increase the expression level of the p-MLC.These studies have shown that the p-MLC changes in cells by different signaling pathways and regulates cell pathophysiological process. After Cerebral hemorrhage, the release of the hemoglobin may be the major pathological media closely connected to the destruction of the blood-brain barrier However, at present there is little report about effects of endothelial cells on hemoglobin Rho kinase blood brain barrier after cerebral hemorrhage. Therefore, the study in vivo by injecting hemoglobin to in SD rat brain resulted in the dynamic detection of endothelial cells of the blood-brain barrier, Rho kinase Ⅱ phosphorylation of myosin light chain (p-MLC) and the changes of expression of tight junction protein claudin-5. In vitro experiments in human umbilical vein endothelial cells (HUVECs), different concentrations of hemoglobin were added respectively, then observing in different time the changes of the permeability,the expression changes of tight junction protein ZO-1, F-actin and p-MLC. By experiments in vivo in in vitro, to further explore the pathophysiological mechanism of Hb causing tight juction damage after cerebral hemorrhage, explaining the occurrence of encephaledema, development of mechanism which provide a new theoretical basis for the future treatment.Objective:In this study of culturing HUVEC line in vitro,.we added a final concentration of5umol/L,20umol/L and40umol/L hemoglobin respectively. In3h,6h and12h, We detected the permeability of monolayer endothelial cells, phosphorylation of myosin light chain, actin F-actin and the expression changes of tight junction protein ZO-1. In vivo injecting hemoglobin to SD rat brain results in dynamic detection of the endothelial cells of the blood-brain barrier, Rho kinase Ⅱ phosphorylation of myosin light chain (p-MLC) and the changes of expression of tight junction protein claudin-5. It provides a theoretical basis for the further study of hemoglboin causing BBB tight junction damage and explains the molecular basis and pathophysiological significance of hemoglobin causing BBB tight junction damage.Methods:1. To observe the proliferation of HUVEC cell growth and draw growth curve of cell by ordinary inverted microscope. To identify the cells by immunohistochemical methods Ⅷ factor.2. To judy hemoglobin permeability changes of a single layer of endothelial cells by detecting the amount of FITC-dextran of the Transwell outer pool. According to the final concentration of the hemoglobin of5umol/L,20umol/L and40umol/L respectively, the Transwell within pool growth monolayer HUVEC was stimulated. The Transwell outside pool FITC-dextran content was detected in3h,6h and12h; without hemoglobin as a comparing group.3. To observe the expression changes of the tight junction protein ZO-1and actin proteins F-actin by immunofluorescence. Owned hemoglobin with a final concentration of5umol/L、20umol/L and40umol/L stimulates monolayer of endothelial cells for6h to observe the expression changes of tight junction protein ZO-1and actin F-actin,4. To detect the expression changes of tight injunction protein ZO-1by Western Blot method. The hemoglobin with a final concentration of5umol/L、20umol/L and40umol/L stimulates the Single layer of endothelial cells for6h, without adding hemoglobin as comparing group, to detect the expression changes of tight junctions ZO-1.5. To detect the expression changes of myosin light chain and phosphorylated-myosin light chain by Western Blot method. The hemoglobin with a final concentration of20umol/L stimulates monolayer HUVEC. To detect the expression changes of the myosin light chain and phosphorylated-myosin light chain in1h,3h,6h and12h respectively. 6. Rats were randomly divided into normal control group and hemoglobin group. According to the successful establish of the model after different time points of death, the hemoglobin group were divided into24h group,48h group,3d group and7d group. Through injecting Hb into rat brain, the immunohistochemical method examined Rho kinase II around hematoma and the expression changes of p-MLC, the Evans blue method detected the changes of the permeability of blood brain barrier, the immunofluorescence method and mRNA detected the expression changes of tight junction protein claudin-5of the blood brain barrier.7. Statistical methods:Data are presented as mean standard deviation (±S) and SPSS19.0statistical software for analysis. One-way ANOVA was used in group comparison. For the homogeneity of variance, use the F-test; For the Heterogeneity of variance. Use the Brown-Forsythe approximate F-test method. Tamhane’s T2was used in comparison of multiple groups. When difference is P≤0.05, the research is statistically significant.Results:1.Observe and count the digestion of cell HUVEC by the. ordinary inverted microscope, and draw the process of cell growth and proliferation:cell digestion,suspension, complete adherence after6hours of inoculation; In the first day of cell growth, the cells were completely adherent stretch, in a form of circular, triangular, polygonal, irregular; In the second day of cell growth, the cells were in quiescent, i.e no cell proliferation. In the third day of cell growth, cell proliferation increased significantly, cell volume became larger and cytoplasm was abundant, the cell density of2or more nuclei can reach to above80%; In the fourth day of cell growth, the cell density increased further, the cell volume became smaller, no significant cell gap appeared, its appearance came to typical cobblestone, the cell density arrived to95-100%; In the fifth day of cello growth, the increase in cell density was not obvious and the cells looked similar to the fourth day; In the seventh or eighth day, the cells grew old gradually.2.The Immunohistochemistry method identified that HUVEC cell lines belong to the endothelial cells, the cytoplasm came with brownish yellow or brown particles, In PBS negative comparison, no DAB color happened.3.To evaluate the change of the permeability of endothelial monolayers by detecting the content of the FITC-dextran outside of Transwell pool. The relative permeability of the control group was1.017+0.100; When the concentration was5umol/L, after3h,it was1.107+0.111; after6h,it was1.200+0.221and after12h it was1.977+0.140; When the concentration was20umol/L,it was1.357+0.076.3.343+0.153、4.983+0.230after3h,6h and12h respectively; While when the concentration is40umol/L,it was1.528+0.698.4.480+0.178and6.670+0.481. Compared with the control group, When the concentration was5umol/L, the content of the FITC-dextran increased after12h; It increased gradually under20umol/L after6h; While it increased obviously under40umol/L after3h. The difference was statistically significant (P<0.05). These results show that the role of Hb monolayer endothelial cells showed a time-and concentration-dependent effect.4. To observe express changes of the cell tight junctions Protein ZO-1and actin F-actin by Immunofluorescence method:For the control group, the protein ZO-1in monolayer for connecting mesothelial cells mainly distributed in cells near the cell membrane, continuous compact arrangement, and smooth edge. It had some results when different concentrations of hemoglobin stimulated monolayer endothelial cell for6hours:Comparing with the control group, the cell tight junctions protein ZO-1did not change much when the final concentration of hemoglobin was5umol/L. The cell tight junctions protein ZO-1decreased and broken, and the cell gap became larger when the final concentration of the hemoglobin was20umol/L. The express of ZO-1decreased obviously, cell gap became larger or even broken and with a jagged form around when the Hb with a final concentration of40umol/L. The endothelial cells F-actin of the control group mainly distributed in surrounding cells, the area of the cell junctions was dense, and inside of the cell, there was little F-actin; The F-action mainly distributed in cells and arrangement was disordered when the Hb was stimulated with final concentration40umol/L.5.To detect the express changes of tight junctions protein ZO-1by Western Blot method. To judy the express content of tight junctions protein ZO-1by the ratio between O-1/β-actin and the express changes of tight junctions protein ZO-1when different Hb final concentration stimulated monolayer endothelial cells for6h. The tight junctions protein ZO-1did net change obviously when the Hb final concentration was5umol/L. There was no statistical mean at all when comparing with the control group. While the express of tight junctions protein ZO-1changed greatly when the Hb final concentration was20umol/L and Oumol/L. There was statistical mean when comparing with control group(P<0.05).The results showed that when the Hb final concentration was higher, the express of the tight junctions protein ZO-1was lower.6.To detect the express changes of Phosphorylated myosin and myosin by Western Blot method. To judy by the ratio between p-MLC and MLC and observe the express changes of p-MLC in1h,2h,6h and12h when stimulating the monolayer endothelial cells with Hb final concentration20umol/L. The p-MLC has no obvious change when Hb stimulated the monolayer endothelial cells for1h. The difference has no statistical mean when comparing with control group; The content of P-MLC express increased when the Hb final concentration is20umol/L for stimulating3h.As time went on, the content of express increase gradually. The difference has a statistical mean when comparing with control group(P<0.05).7.To observe the result of Rho kinase Ⅱ and the expression changes of p-MLC by immunohistochemical staining in brain tissue of rats:The result of expression changes of Rho kinase Ⅱ:In the normal control group, there was no expression of Rho kinase Ⅱ in the endothelial cells of the blood-brain barrier. For the hemoglobin group, there was a little brown granules in24h in hematoma side blood-brain barrier endothelial cells; The expression of Rho kinase Ⅱ increased in48h and3d, which mainly expressed in side endothelial cells of the blood-brain barrier of Hematoma, with a form of Brown or brownish yellow granules. As time went on, the expression of Rho kinase Ⅱ decreased in7d. The result of expression changes of P-MLC showed:In the normal control group of brain tissue, there is little expression of blood brain barrier endothelium p-MLC and distributed in a low level of brown line shape mainly around blood vessels. For the hemoglobin group, there is little expression of P-MLC in24h, the expression of hematoma blood-brain barrier endothelial cell p-MLC increased in48h and3d, distributing in the brown line shape. The expression decreased obviously in7d.8.To assess the effect of hemoglobin on the permeability of blood-brain barrier by the application of the Evans Blue permeability experiments. Compared with the normal control group, the content of the rat brain Evans blue was2.99±1.45(ug/g), the hemoglobin group was8.91±2.11,7.26±2.18,11.57±3.13,6.55±1.07(ug/g) in24h,48h,3d and7d. Compared with the normal group, the content of Brain tissue Evans blue was significantly higher in the Hb group.The difference was statistically significant.9.To detect the expression changes of BBB tight junction protein claudin-5by tissue immunofluorescence method and quantitative real-time mRNA method: Immunofluorescence results are shown:As for rats in the normal control group, the expression of tight junction protein claudin-5in endothelial cells of the blood-brain barrier was clear and strong, distributing in a a continuous strip. As for the hemoglobin group, the expression of the blood-brain barrier tight junction protein claudin-5around hematoma in endothelial cells decreased, it arrived to the lowest point in48h and3d and recovered gradually at in7d. The relative expression results of blood-brain barrier tight junction protein claudin-5mRNA are shown:Compared with the normal group1.09±0.15, the hemoglobin group showed0.26±0.13、0.31±0.08、0.21±0.04、0.19±0.09respectively in24h,48h,3d and7d; Compared with the normal group, the expression level of the Blood-brain barrier tight junction protein claudin-5mRNA decreased for the hemoglobin group when it was in24h,48h,3d and7d. The difference was statistically significant (P<0.05).Conclusion:1. Hemoglobin can destroy tight junctions of monolayer endothelial cells and increase permeability, which can lead to dysfunction of endothelial barrier.2. Hemoglobin can increase the express of monolayer endothelial cells, which can lead to the formation of a large number of stress fiber in cells F-actin and the decrease of express of tight junctions ZO-1in cell gaps. This change may be the main pathological and physiological process in the destroy of endothelial cells tight junctions.3. Hemoglobin can make the brain tissue around hematoma in rats with the increased expression of Rho kinase Ⅱ. On one hand, it can cause the increased expression of p-MLC and tight junction damage leading to increasing in permeability of blood brain barrier; On the other hand, it can directly lead to the tight junction protein claudin-5phosphorylation, its expression decreased and blood-brain barrier damage. The changes may be one of the most important mechanisms leading to the occurrence and development of the vasogenic brain edema after cerebral hemorrhage. |
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